JPH0626667B2 - Cyclodextrin adsorbent and its application - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a cyclodextrin adsorbent, and more particularly, to a cyclodextrin-containing solution, for example, a cyclodextrin-producing reaction mixture containing an unreacted raw material and various byproducts. The present invention relates to a cyclodextrin adsorbent capable of adsorbing with high efficiency and high selectivity and its use.

Cyclodextrin is usually produced by treating starch with cyclodextrin glycosyl tranferase (CGTase), in which case the reaction product mixture contains unreacted cyclodextrin as well as unreacted product. Starch and various degradation products of starch, such as oligodextrin, glucose, and enzymes used, are mixed. Formation of cyclodextrin containing such unreacted raw materials and various by-products As one method for separating cyclodextrin from a reaction mixture, an organic solvent that can be included by cyclodextrin is added to the reaction mixture. A method has been proposed in which cyclodextrin is precipitated as a solid and fractionated.
The use of such an organic solvent is not preferable for the main applications of cyclodextrin in the fields of medicine and food, and development of a separation method without using an organic solvent is desired.

As a method for separating and purifying cyclodextrin without using such an organic solvent, conventionally, (1) a method of adsorbing the resin on a porous styrene-divinylbenzene copolymer (JP-A-56-805), (2) A method of adsorbing and removing only reducing sugar from a mixed solution of reducing sugar and cyclodextrin using an anion exchange resin to separate cyclodextrin (JP-A-51-136,889), (3) strong acid cation exchange resin A method in which an alkali metal salt or an alkaline earth metal salt is packed and treated in a column to fractionate into a cyclodextrin compartment and a glucose compartment (JP-A-57-
However, the porous resin and the ion exchange resin used in these conventional methods have poor selectivity and require a complicated elution treatment when separating and recovering cyclodextrin. Is not completely satisfactory.

Therefore, the present inventors have completed the present invention as a result of earnest research on an adsorbent having excellent selectivity and capable of separating cyclodextrin by a simple elution operation.

Therefore, according to the present invention, a ligand having a size that can be included by cyclodextrin is a spacer group (space group).
There is provided a cyclodextrin adsorbent characterized by comprising a water-insoluble resin substrate chemically bound via (r radical).

Hereinafter, for the cyclodextrin adsorbent of the present invention,
It will be described in more detail.

Ligand: A feature of the present invention is as a cyclodextrin adsorbent,
Instead of the conventional porous resin or ion-exchange resin, a resin substrate in which a ligand having a size to be included by cyclodextrin is bound via a spacer group is used.

Thus, the ligand used in the adsorbent of the present invention enters the cyclodextrin annular cavity,
It is an atomic group of a size that can be adsorbed to the inner surface of the cavity by a hydrophobic bond, that is, a size that can be included, and generally includes a hydrophobic organic residue having a bulky molecular structure. It The size of the ligand clathrated by the cyclodextrin is limited by the diameter of the cyclodextrin's annular cavity, and when the size of the ligand is considered as an ellipsoid just surrounding the entire atomic group of the ligand,
The minor axis of the ellipsoid is about 4 to about 10Å, preferably about 5
A size that is within the range of about 7.5Å is suitable.

Therefore, specific examples of the ligand that can be used in the adsorbent of the present invention include the following: tert-
Butyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-C ₃ -C ₇ such dimethylbutyl, preferably C ₄ -C ₆ branched-chain aliphatic hydrocarbon group; cyclohexyl, cyclohexenyl , adamantyl, norbornenyl, glycyrrhetinic acid residue, Kanfua residue, borneol residue, 3-dimethyl-cyclohexyl, 4-ethylcyclohexyl, abietic acid residue, hecogenin residue, C ₅ -C _34, such as Sutebio acids residues, preferably Is a C ₆ -C ₂₉ alicyclic hydrocarbon group; aromatic hydrocarbon groups such as phenyl, naphthyl, p-tert-butylphenyl, and fennathryl;
Heterocyclic groups such as furyl, thiophene residue, tetrahydrofuryl, tetrahydrothiophene residue.

Preferred among these ligands are tert-
Butyl group, cyclohexyl group, adamantyl group, norbonenyl group, glycyrrhetinic acid residue, abietic acid residue,
A p-tert-butylphenyl group and the like can be mentioned.

Water-insoluble resin substrate: The resin used as the substrate in the adsorbent of the present invention is
There is no limitation on the kind as long as it is substantially insoluble in water, and any one can be used. Hereinafter, representative ones of the resins that can be used as the base material of the adsorbent of the present invention are exemplified, but the resin base material for the adsorbent used in the present invention is not limited to these and will be described later. It should be understood that any resin can be used as long as the method allows the ligand to be introduced via a spacer group.

In the present specification, the “resin substrate” refers to a skeleton structure portion which is a main component of the resin, and when the resin has a functional group-containing side chain for introducing a ligand, the side chain is excluded. It is a part.

It should be understood that in the following, not only such a resin substrate but also a resin substrate having a functional group-containing side chain for introducing a ligand has been already introduced.

(1) Polysaccharide-based compounds and derivatives thereof Polysaccharide-based compounds include, for example, cellulose, dextran, agarose, chitin, chitosan, and the like, and its derivatives include cross-linked products of the polyheterogeneous compound or Those in which a side chain containing a functional group such as an epoxy group, an activated carboxyl group, a haloacetyl group, a tertiary or quaternary ammonium salt group is introduced by utilizing a hydroxyl group existing in a multi-class compound are included. [For the introduction method of such a group, see, for example, "New Experimental Chemistry Course", Vol. 20, No. 67-70.
Page, published by Maruzen Co., Ltd. on June 20, 1978; Cautrec
asas, J, Biol. Chem., 245 3059 (1970);
P. Cautrecasas, I. Parikh, Biochemistry, 11 , 2
291 (1972); Sundberg, J .; Porath, J .; C
hromatgr. , 90 , 87 (1974)].

Specific examples of such multi-class compounds and derivatives thereof include AH-Sepharose 4B and CH-Sepharose 4
B, epoxy-activated cepharose 6B, diethylaminoethyl-cephadex, quaternary aminoethyl-cephadex, sulfopropyl-cephadex, and the like, which are commercially available from Huarmacia Huaine Chemical Co., and products such as chitopearl and chitobeads. Examples include those commercially available from Fuji Spinning Co., Ltd.

(2) Styrene-based resin A styrene-based resin is a styrene-based resin that contains styrene or a nuclear-substituted derivative thereof (for example, haloalkyl-substituted styrene, aminoalkyl-substituted styrene, quaternary aminoalkyl-substituted styrene, carboxyalkyl-substituted styrene) as a main constituent unit. Or a copolymer, especially a chloromethylated polystyrene, a styrene-divinylbenzene copolymer or a functional group-containing side chain such as a chloromethyl group, an aminoalkyl group, a quaternary aminoalkyl group or a carboxyalkyl group on its benzene ring. Those introduced with are preferable.
Specific examples of such resins include those commercially available from Mitsubishi Kasei Kogyo Co., Ltd. under the trade names of chloromethylated polystyrene, Diaion WA resin, Diaion PA resin, Diaion WK resin and the like. Can be mentioned.

(3) Polyacrylamide-based resin As the polyacrylamide-based resin, a homopolymer of acrylamide or a copolymer mainly composed of acrylamide,
Alternatively, through the amide group of these polymers, an aminoalkyl group, a quaternary aminoalkyl group, an aminophenyl group, a quaternary aminoalkyl group,
Those containing a primary aminophenyl group and the like are included, and specific examples thereof include those commercially available from Bio-Rad Co. under the trade names of Bio-Gel, Enzacryl and the like.

(4) Peptides such as p-amino-DL-phenylalanine-L-leucine.

(5) Others Various kinds of pendant side chains having a functional group capable of chemically binding a ligand by the method described below or having been introduced
(Co) polymers, for example, used for producing a water-insoluble resin known per se, homopolymers of various monomers or copolymers composed of a suitable combination thereof, and specific examples include Polymers of dienes such as butadiene, butadiene and isoprene; Polymers of vinyl ethers such as chloromethyl vinyl ether and allyl glycidyl ether; Polymers of acrylic acid such as acrylic acid amide, acrylic ester, acrylonitrile and N-methylol acrylamide; Methacryl Acid ester, methacrylonitrile, glycidyl methacrylate, 2-hydroxyethyl methacrylate, 2-sulfoethyl methacrylate,
Polymers of methacrylic acids such as 2-chloro (bromo) ethyl methacrylate; polymers of unsaturated carboxylic acids such as itaconic acid and crotonic acid; polymers of phenol, tetraethylenepentamine, and phenylenediamine with formalin; and The copolymer which consists of those 2 or more types of monomers can be mentioned. Further, in the case of various copolymers using a derivative obtained by binding the above ligand to these monomers by any one of the following covalent bonding methods, the carrier of the present invention does not need to be bound with the following ligands. Can be Incidentally, these polymers can be produced by a polymerization method known per se, for example, radical polymerization or polycondensation method.

Among the resins described above, preferable examples include the polysaccharide compound (1) and derivatives thereof, and among them, chitosan and its crosslinked product are particularly preferable.

Production of Cyclodextrin Adsorbent According to the Present Invention The adsorbent of the present invention can be produced by introducing the above-mentioned ligand into the above water-insoluble resin substrate via a suitable spacer group. The method will be described below.

(1) Method using covalent bond reaction In the formula, is a water-insoluble resin substrate, L is a ligand, X and Y represent functional groups capable of reacting with each other to form a covalent bond, Z ₁ represents a spacer group, and Q ₁ represents a spacer group Z. _{1 is} a part of ₁ and represents a portion excluding the functional group X from the functional group-containing side chain that the water-insoluble resin has or is introduced into the resin, and Q ₂ represents another part of the spacer group Z _1. It represents a divalent residue that connects the ligand L and the functional group Y, and p and q are 0 or 1, respectively.

The functional groups represented by X and Y and the type of reaction used for the covalent bond thereof are shown in Table 1 below: In Table 1 above, the reactive derivative of the carboxyl group (—COOH) is a carboxylic acid halide (eg, —COOH).
Cl, --COBr), carboxylic acid anhydride, mixed acid anhydride,
Active carboxylic acid ester group Etc. [the same applies to Table 2 below].

Further, the formulas (I) and (II) used in the above reaction scheme A are
Examples of the starting material and the type of the adsorbent resin of the formula (III) are as follows.

(i) Or Or Or (ii) In the formula, Represents a water-insoluble resin substrate in which hydroxyl groups are partially omitted in the basic bone nucleus of cellulose.

In addition, here And L are No. 1 to 18 and No. 33 to in Table 2 above.
41's And the same as L, Corresponds to the spacer group.

(iii) In the formula, Represents a water-insoluble resin substrate in which hydroxyl groups are partially omitted in the basic bone nucleus of crosslinked dextrin.

In addition, here And L are No. 1 to 18 and No. 33 to in Table 2 above.
41's And the same as L, Corresponds to the spacer group.

(iv) Styrenic resin a) In the formula, is, for example, the general formula Represents a styrene resin substrate represented by.

here, And L are No. 1 to 18 and No. 33 to in Table 2 above.
41's And the same as L, Corresponds to the spacer group.

b) In the formula, is, for example, the general formula Represents a styrene resin substrate represented by.

here, And L are those of Nos. 19 to 31 in Table 2 above. And the same as L, Corresponds to the spacer group.

c) In the formula, is, for example, the general formula Represents a styrene resin substrate represented by, and represents m = 5 to 20.

here, And L are No. 1 to 18 and No. 33 to in Table 2 above.
41's And the same as L, Corresponds to the spacer group.

(v) Acrylic resin a) Where is a general formula Represents an acrylic resin substrate represented by.

here, And L are those of Nos. 19 to 31 in Table 2 above. And the same as L, Corresponds to the spacer group.

b) Where is a general formula Represents an acrylic resin substrate represented by.

here, And L are Nos. 1-18 and Nos. 33-4 in Table 2 above.
One And the same as L, Corresponds to the spacer group.

(vi) Polyamino acid resin Where is a general formula Represents a polyamino acid resin substrate represented by.

here, And L are No. 1 to 18 and No. 33 to in Table 2 above.
41's And the same as L, Corresponds to the spacer group.

(2) Method using ionic bond reaction Wherein, and L have the above-mentioned meanings, A and B represent functional groups capable of reacting with each other to form an ionic bond, Z ₂ represents a spacer group, and Q ₃ represents a part of the spacer group Z ₂ . Which represents a portion of the functional group-containing side chain that the water-insoluble resin has or has been introduced into the resin, excluding the functional group A, and Q ₄ is another part of the spacer group Z ₂ . It represents a divalent residue connecting the ligand L and the functional group B, and a and b are 0 or 1, respectively.

A specific example of the reaction formula B is as follows.

(a) (b) In the above reaction formulas (a) and (b) Specific examples of L and L include the following.

Water-insoluble resin component of formula (I) or (IV) and ligand of (II) or (V) in the production of the adsorbent of the present invention using the covalent bond or ionic bond reaction shown in (1) and (2) above The reaction with the introduced component can be carried out by a method known per se, for example, “Immobilized Enzyme” edited by Ichiro Chibata, pp. 9-85, March 1975.
Published by Kodansha Co., Ltd. on March 20; K. Mosbach, ed., "Metho
ds in Enzymology ”, Voll44, Academic Press,
New York (1976); Axen, S.M. Ernback, Eu
r. J. Biochem., 18 , 351 (1971) and the like can be carried out according to the method, and therefore the detailed description will be replaced with the citation of this document.

In the adsorbent resin of the present invention, the spacer group interposed between the ligand and the resin substrate prevents the resin substrate from becoming a steric hindrance when the cyclodextrin is in the vicinity of the adsorbent resin and includes the ligand. It is desirable that the spacer group has a length enough to separate the ligand from the resin substrate (support) to a certain extent or more, and the length of the spacer group should be the same as that of the substrate resin and / or the ligand. Although it differs depending on the type and cannot be specified in a general manner, generally speaking, the total length of the spacer base portion and the ligand portion is at least 10 angstroms or more, preferably 10 angstroms or more.
-60 angstrom, more preferably 15-30
Spacer groups can be chosen to have lengths in the Angstrom range.

Here, the total length of the spacer group and the ligand is CP
K (Corey-Pauling Koltun) molecular model (Eal, USA
ing company).

The adsorbent resin provided by the present invention can be molded in any suitable form depending on the application described below,
For example, it can be in the form of beads, fibers, hollow fibers, cylinders, prisms, films, etc., with beads being particularly preferred.

Hereinafter, a specific method for producing the adsorbent of the present invention will be described in more detail with reference to examples.

Example 1 20 ml of dried Diaion WA-20 (OH type) resin was immersed in dry chloroform for 1 hour and then 10.8 ml.
4'-Nitrobenzene-4-tert- dissolved in (77.4 mmol) triethylamine and 50 ml dry chloroform.
Butylbenzoic acid ester 16.2 g (54.1 mmol) was added. After reacting under reflux for 6 hours, remove the resin,
50% (V / V) containing 2% (W / V) sodium hydroxide
It was washed with an aqueous ethanol solution to remove p-nitrophenol. Further, it was washed with water and dried to obtain an adsorbent resin in which the ethyleneamine side chain P-kaist-butylbenzoic acid of WA-20 resin was amide-bonded through its carboxyl group.

Example 2 Using an active ester of 2-norbornene acetic acid, 3-methyl-1-adamantane acetic acid and para-nitrophenol, the same treatment as in Example 1 was carried out to prepare an adsorbent resin having a corresponding ligand.

Example 3 7.9 g (6.8 mmol) of glycyrrhizin are dissolved in 200 ml of dry N, N-dimethylformamide (DMF).

Dicyclohexylcarbodiimide (DCC) 7.0
A solution of g (33.9 mmol) dissolved in 100 ml of dry DMF was added, and the mixture was stirred at room temperature for 3 hours.

20 ml of dried Diaion WA-20 (OH type) resin was added, and the mixture was stirred at room temperature for 24 hours.

After the reaction, the resin was taken and thoroughly washed with ethanol to remove dicyclohexylurea. Wash with water and dry WA-2
An adsorbent resin was obtained in which kerithyrrhizinic acid was amide-bonded to the ethyleneamine side chain of No. 0 resin via its carboxyl group.

Example 4 To 20 ml of dried Diaion WA-20 (OH type) resin was added 30 ml of dried tetrahydrofuran and 20 ml (0.14 mol) of triethylamine. 17 ml (0.127 mol)
A solution of cyclohexylcarbonyl chloride in 20 ml of tetrahydrofuran was added dropwise under ice cooling in a nitrogen atmosphere, and after the addition was completed, the reaction temperature was raised to room temperature and stirred overnight. Remove the resin, wash away excess reagent with tetrahydrofuran, wash with water, and dry to remove WA-2.
An adsorbent resin in which a cyclohexylcarbonyl group was amide-bonded to the ethyleneamine side chain of 0 resin was obtained.

Example 5 Instead of cyclohexylcarbonyl chloride, myristin chloride or cyclohexylpropionyl chloride was used and treated in the same manner as in Example 4 to obtain an adsorbent resin in which myristyl group or cyclohexylpropionyl group was bonded to WA-20 resin. .

Example 6 30 ml of swollen activated CH Sepharose 4B plus 0.1
After mixing 100 ml of M carbonate buffer (pH 8.0), 22
6 (2.0 mmol) cyclohexylmethylamine was added,
The reaction was carried out at room temperature for 1 hour. Separate the resin, 0.05M Tris buffer (pH 8.0) and 0.05M formate buffer (pH 4. 0).
Alternating washes with 100 ml each of 0) to remove excess cyclohexylmethylamine. Then, it was washed with water and dried to obtain an adsorbent resin in which cyclohexylmethylamine was amide-bonded to the carboxyl group of CH-Sepharose 4B.

Similarly, 1-adamantyloxyethylamine, 4-te
An amine derivative of each ligand such as rt-butylphenylamine was reacted with activated CH sepharose 4B to obtain an adsorbent resin having an agarose-based corresponding ligand.

The resin obtained above is 1630 cm by IR.
Absorption based on carbonyl of amide was confirmed near ^-1 .

Example 7 Dry chitosan resin fully swelled in N, N-dimethylacetamide (hereinafter referred to as DMA) [Chitopearl BCW
1010 (Fuji Spinning Co., Ltd.)] 20 ml DMA 15 ml
Was added, and then a solution prepared by dissolving 2.29 mmol of a mixed acid anhydride of (N-3-cyclohexylpropionyl-β-alanyl) -β-alanine and pivalic acid in 5 ml of DMA was added,
Stir at room temperature for 24 hours. Remove the resin and use acetone 4
Immersion washing with 0 ml was performed 3 times. Furthermore, it was washed with 250 ml of dilute alkaline 50% methanol water and then with 250 ml of water, and substituted chitosan in which (N-3-cyclohexylpropionyl-β-alanyl) -β-alanine was introduced into the amino group of the chitosan resin through an acid amide bond. A resin was obtained.

Example 8 (N-3-cyclohexylpropionyl-β-alanyl)
Instead of -β-alanine, a compound in which β-alanine is linked to 0 to 3 in a chain form by an acid amide bond in cyclohexanepropionic acid, for example, cyclohexanepropionic acid, (N-
3-cyclohexylpropionyl-β-alanyl) -β
-Alanyl-β-alanine or the like, or a compound in which β-alanine is linked to t-butylbenzoic acid by an acid amide bond in a chain form of 0 to 4 groups, for example, (N-4-t-butylbenzoyl-β-alanyl)- β-alanyl-β-alanyl-β-
Alanine or the like or (N-2-norbornaneacetyl-β-alanyl) -β-alanine was used and treated in the same manner as in Example 7 above to prepare a substituted chitosan resin having a corresponding ligand. The resins thus obtained were confirmed by IR to have absorptions at around 1650 and 1550 cm ^-1 based on amide carbonyl.

Example 9 35 ml of PA-308 resin, which was OH type with 4% (wt / V) sodium hydroxide aqueous solution, was packed in a φ15 mm column,
After thorough washing with water, washing with a 50% (V / V) ethanol aqueous solution is performed.

40 g of p-tert-butylbenzoic acid 9.0 g (50.5 mmol)
% (V / V) 50% containing 3.0 ml of methylamine aqueous solution
(V / V) dissolved in 160 ml of ethanol aqueous solution, PA-3
A column of 08 (OH type) resin is flowed at a flow rate of about 65 ml / hr SV. After further washing with about 500 ml of 50% (V / V) ethanol aqueous solution, washing with water and drying, an adsorbent resin in which p-tert-butylbenzoic acid was ionically bonded to PA-308 resin was obtained.

Example 10 Instead of p-tert-butylbenzoic acid, t-butylacetic acid, cyclohexylpropionic acid, glycyrrhizinic acid, adamantylacetic acid, norborneneacetic acid, etc. were used and treated in the same manner as in Example 9 above, respectively. An adsorbent resin having a ligand that reacts was obtained.

Example 11 12.5 ml of QAE A-25 resin made OH type with 1% (wt / V) sodium hydroxide aqueous solution was packed in a column of φ15 mm, thoroughly washed with water, and then with 50% (V / V) ethanol aqueous solution. Perform cleaning.

40% of 5.0 ml (34.5 mmol) of 2-norbornene acetic acid
(V / V) 32% containing 1.0 ml of methylamine aqueous solution (V
/ V) Dissolve in 125 ml of ethanol aqueous solution and use QAE A
A column of -25 (OH type) resin is flowed at a flow rate of about 65 ml / hr SV.

After that, about 300 ml of 50% (V / V) ethanol aqueous solution
After washing with water, washing with water and drying, an adsorbent resin in which 2-norbornene acetic acid was ionically bonded to the anion exchange part of the QAE A-25 resin was obtained.

Example 12 Similarly, 3-methyl-1-adamantane acetic acid, abietic acid, myristic acid, cyclohexanepropionic acid and the like were ionically bonded to obtain a corresponding adsorbent resin.

Cyclodextrin Adsorption Properties of the Adsorbent Resin of the Present Invention Regarding the adsorbent resin of the present invention, its cyclodextrin (hereinafter abbreviated as “CD”) adsorption properties are shown in Table 4 to
11 shows. In the adsorption test, 35 ml of each CD adsorbent carrier shown in each table was packed in a column (1.5 cm × 25 cm), and the carrier was thoroughly washed with water, and then the CD-containing liquid [α-CD, 1 (W / V)
%; Β-CD, 1 (W / V)%; γ-CD, 1 (W / V)% aqueous solution] 45 ml. Then rinse with 350 ml of water and wash with 180 ml of ethanol / ice (1/1) mixture.
The content of each CD in the flow-through was measured by HPLC (Shodex, RS-p
ak CD-613 _{column; CH 3 CN / H 2 O} (65/3
5), 15 ml / min) method. Values obtained by converting the obtained CD amount into the CD amount (g) adsorbed per resin 1 are shown in Tables 5 to 9.

Table 11 shows the amount of β-CD adsorbed on the adsorbent resin corresponding to the change in the length of the spacer group. The converted value of the adsorption amount was calculated by the same method as above.

Then the general formula When manufacturing the resin represented by And the amount of piparinic acid anhydride charged and β to the resin
-Table 11 shows the correlation of the CD adsorption amount.

Use of the Adsorbent Resin of the Present Invention Since the cyclodextrin-adsorbing carrier of the present invention has the selective physical adsorption ability between the ligand and the hydrophobic cavity of cyclodextrin, as described above, the following specific applications are possible. is there.

(1) Selective Separation and Purification of Cyclodextrin from Cyclodextrin-Containing Aqueous Liquid The adsorbent of the present invention is, for example, cyclodextrin (C
D) It can be used to selectively separate and purify CD from the production reaction solution. As described above, CD is an α-, β- and / or γ-CD produced by reacting various CGTases with an aqueous suspension or aqueous solution of starch, unreacted starch and various oligodextrins and glucose produced as by-products. It is produced by separating and purifying CD from a reaction solution containing (see the above-mentioned publication). The adsorbent of the present invention is
Since it can selectively adsorb CD, it is used as a carrier for column chromatography, or it is contacted with a cyclodextrin-containing aqueous liquid such as the above reaction liquid by a batch method so that CD is adsorbed onto the adsorbent. Then, after separating unreacted starch and by-products, the CD adsorbed from the adsorbent is desorbed by using hot water or an alkanol / water mixed solvent, whereby the CD can be separated and purified in good yield. .

The contact between the adsorbent and the cyclodextrin-containing aqueous liquid can generally be carried out at room temperature, but in the case of about 30 to about 7
The heating may be performed at 0 ° C. The contact time is not strictly limited, but is usually in the range of 1 to 4 hours.

The temperature of the hot water used for the desorption of cyclodextrin from the cyclodextrin-adsorbed resin may be in the range of about 70 to about 90 ° C, and the alkanol in the alkanol / water mixed solvent may be methanol, ethanol. , Lower alkanols such as propanol, etc., and the mixing ratio of alkanol / water is generally 1 /
The range of 5 to 3/5 (V / V) is preferable. In addition to the above, acetone, acetonitrile, tetrahydrofuran, dioxane, etc. can also be used for desorption welding.

Examples of the cyclodextrin-containing aqueous liquid that can be used in the above method include, in addition to the cyclodextrin-forming reaction liquid described above, a chemical reaction liquid or CD using CD as a catalyst.
An enzyme reaction solution or a microbial fermentation solution in which is used as a protective agent for the product is exemplified.

The specifics of the selective separation and purification method of cyclodextrin are shown below.

Example 1 100 g of horseradish starch with neospitase (Nagase Sangyo Co., Ltd.)
(Manufactured) was added and then water was added to bring the total volume to 2, and 80
After heating and liquefying at 20 ° C. for 20 minutes, it was autoclaved at 120 ° C. for 10 minutes. Then cool to 50 ° C. and Bacillus ohbensis FERM P-199
CGTase1000u obtained from 0 was added to pH 6.0, 5
The CD generation reaction was performed at 0 ° C. for 24 hours. 1 reaction mixture
After inactivating the enzyme by heat treatment at 00 ° C for 15 minutes, a part of the enzyme was measured for the amount of produced CD by liquid chromatography. Α-CD was 0.8% (wt / wt), β-CD 3 CDs
It was 1.0% (wt / wt) and γ-CD was 8.5% (wt / wt).

Next, this reaction solution was passed through a column filled with 700 ml of PA-308 ion-bonded with tert-butylacetic acid by a descending method, and then the column was washed with 3.0 water. When 50% (V / V) ethanol aqueous solution 5 was passed through this column and concentrated to dryness, β-CD3 having a purity of 95.3% was obtained.
2.0 g was obtained.

Further, the column solution of the reaction solution and the washing solution were mixed, and after passing through a column filled with 500 ml of PA-308 ion-adsorbed with glycyrrhizin by a descending method, the column was washed with water of 2.0.

When 70 ° C. hot water 4 was passed through this column and the mixture was concentrated to dryness, 6.7 g of γ-CD having a purity of 98.0% was obtained.

Example 2 425 ml of the reaction solution treated in the same manner as in Example 1 (measured values of various CDs: 7.40 g of .beta.-CD, 1.36 g of .gamma.-CD) were added to (N-3
-Cyclohexylpropionyl-β-alanyl) -β-
The solution was passed through a column filled with 100 ml of chitosan resin bound with alanine at 44 mol% (per constituent glucosamine) by a descending method. After washing the column with 400 ml of water, 50%
The adsorbed CD was eluted by passing 755 ml of (V / V) ethanol aqueous solution. The eluate was concentrated to give a β of 99.9% purity.
5.87 g of -CD crystals (recovery rate 83.4%) was obtained.

(2) Increase in yield of specific CD during CD production reaction The CD production reaction is an enzymatic reaction by CGTase as described above, and reversibly proceeds. Therefore, in order to increase the yield of CD, an organic solvent capable of being clathrated by CD is allowed to react in the reaction system to cause the reaction to remove the produced CD as a precipitate outside the reaction system, and to convert the raw material starch etc. into CD. It is known that the rate can be increased (for example, JP-A-60-1563).
No. 98).

By the way, the adsorbent resin of the present invention is used as a ligand for CD
Since it has an organic residue that can be clathrated by the above, by allowing the carrier to coexist in the reaction system, the conversion rate can be increased, and by selecting a ligand, a desired α, β or γ-CD can be selected. The production rate of CD can be increased, and since the produced CD is selectively adsorbed on the adsorbent resin, high-purity CD can be easily obtained from the resin by the above-mentioned CD adsorption method.

The reaction for producing CD using the adsorbent resin of the present invention is carried out under the reaction conditions known per se using starting materials known per se, except that the production reaction is carried out in the presence of the adsorbent resin. [E.g., British Patent 1,459,654; U.S. Pat. No. 4,31.
7,881 Specification; Agricultural and Biologica
Chemistry, 49 , 1189-1191 (1985)]. For example, soluble starch is usually used as a starting material, and CD is produced by subjecting this to an enzymatic reaction with CGTase in an aqueous medium. Examples of CGTase include Bacillus macerans, B. circulans, B.I. megate
rium, B. ohbensis, Klebsiella preumoniae, slkalopb
ilie Bacillu, Micrococcus varians, M.I. Those derived from luteus or the like can be used, and the enzyme reaction can be completed in about 12 to 48 hours at a temperature of 30 to 65 ° C. under an optimum pH condition corresponding to each enzyme.

According to the present invention, the CD production reaction by this enzymatic method is carried out in the presence of the adsorbent resin described above, whereby the yield of produced CD can be increased by about 50 to 150% as compared with the conventional method.

Examples of the method for carrying out the reaction for producing the CD in the presence of the adsorbent resin include, for example, charging the adsorbent resin in the form of beads into a reaction vessel for the CD, and batchwise or continuously while stirring and suspending. A method of carrying out an enzymatic reaction on; a reaction column filled with an adsorbent resin molded in a suitable form such as beads,
Examples include a method of conducting the above-mentioned aqueous solution containing the starting material and CGTase once or several times to conduct electricity.

One advantage of the above-described CD-forming reaction according to the present invention is that it can be carried out with higher concentrations of soluble starch than in conventional methods. For example, in the conventional CD-forming reaction, the soluble starch concentration is 5 to
7% (wt / wt), but using the method of the present invention, CD
The starch concentration can be increased to about 10 to about 20% (wt / wt) without substantially reducing the production efficiency.

Although the aqueous solution concentration of starch used in the present invention is not particularly limited, it can be selected within the range of 3 to 20 (wt / wt)%, preferably 5 to 15%.

The optimum reaction temperature is different depending on the type of CGTase used and is not critical.
When using CGTase obtained from ohbensis, 45 to 6
The temperature may be 5 ° C, preferably 50 to 60 ° C, and the pH of the reaction system may be 5 to 11, preferably 6.5 to 7.5.

The reaction time depends on the reaction time, the reaction mode, the type of CGTase used, and the amount used, and it cannot be strictly limited, but the reaction may be continued until the starch used as the substrate raw material is almost consumed.

Further, the amount of the resin of the present invention used should be equal to or more than the amount of the resin capable of sufficiently adsorbing the CD when it is assumed that the starch used as the substrate raw material is completely converted into CD regardless of the reaction mode. You can The adsorbent resin C
The amount of D adsorbed was determined by a simple experiment (routi
It can be determined empirically by a ne experiment).

According to the method of the present invention described above, the generated CD can be directly recovered from the reaction system in the form of being adsorbed on the adsorbent resin, so that complicated CD separation as in the conventional method,
No purification operation is required, which is extremely advantageous industrially.

Next, the CD formation reaction will be described more specifically with reference to Examples.

Example 3 Soluble starch (10 g) was added to warm water to bring the total volume to 500 ml, and two reaction solutions were prepared by autoclaving at 120 ° C. for 15 minutes.

Then, cool to 55 ° C, add 100 units of CGTase obtained from Bacillus ohbensis to each, and add 1 to pH 7.0.
The CD generation reaction was performed for 9 hours. To one of the reaction solutions, 50 ml of a resin of PA-308 to which p-tert-butylbenzoic acid had been ion-adsorbed was added 3 hours after the start of the reaction.

After 19 hours of the reaction, the CD production amount of each reaction solution was determined by liquid chromatography. Separate the resin from the reaction solution containing the resin, wash the supernatant and the resin thoroughly with water, then add 50%
The eluate obtained by eluting the CD adsorbed with the (V / V) ethanol aqueous solution was separately determined by liquid chromatography.
The results are shown in Table-12.

Example 4 5 g of horseradish starch and neospitase [manufactured by Nagase & Co., Ltd.]
Water was added to 3 mg to bring the total volume to 50 ml. After liquefaction at 80 ° C for 20 minutes, autoclave treatment was performed at 120 ° C for 15 minutes. Cool to 50 ° C., add 3 g of salt, (N-
3-cyclohexylpropionyl-β-alanyl) -β
-Alanine 44 mol% (per component glucosamine) bound to Chitopearl BCW1010 60 ml resin, Bacillu
Add 250 units of CGTase obtained from Sohbensis,
19 hours C with gentle stirring at 7.0 and 50 ° C
The D formation reaction was performed. After the reaction, the resin is separated and water 10
After washing with 0 ml, 50% (V / V) ethanol aqueous solution 500
The CD adsorbed in ml was eluted. The amount of CD produced was determined by measuring the reaction supernatant, the washing solution, and the eluate by liquid chromatography. The results are shown in Table 13 below. Further, as a control, a CD generation reaction without resin was carried out.

Example 5 Substrate solution liquefied under the same conditions as in Example 4 (concentration 10% (wt / w
t)) Add 50 units of CGTase to 50 ml, and
Reacted for 5 hours. After that, while continuing the reaction at 50 ° C,
The reaction solution was added to (N-3-cyclohexylpropionyl-β
-Alanyl) -β-alanine 44 mol% (per constituent glucosamine) was passed through a column (diameter 3 cm) packed with 42 ml of resin bound to Chitopearl BCW1010, and circulated for 18 hours with the reaction tank (flow rate 1.5 ml). / Min). After the reaction, the resin was washed with 80 ml of water, and the adsorbed CD was eluted with 350 ml of 50% (V / V) ethanol aqueous solution. The CD production amount was determined in the same manner as in Example 4. As a control, a similar reaction was performed using a column not filled with resin. The results are shown in the table below.
14 shows.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Koki Fujii 4-5-1-403 Konandai, Konan-ku, Yokohama-shi, Kanagawa Prefecture (72) Tomoyuki Ishikura 22-32 Owada, Chigasaki-shi, Kanagawa Prefecture

Claims (15)

[Claims] 1. A cyclodextrin adsorbent comprising a water-insoluble resin substrate to which a ligand having a size capable of being included by cyclodextrin is chemically bound via a spacer group. 2. The size of the ligand is such that, when conceived as an ellipsoid that just surrounds the entire atomic group of the ligand, the minor axis of the ellipsoid falls within the range of about 4 to about 10Å. The adsorbent according to claim 1. 3. A ligand selected from a C _{3 to} C ₇ branched aliphatic hydrocarbon group, a C _{5 to} C ₃₄ alicyclic hydrocarbon group, an aromatic hydrocarbon group and a heterocyclic group. The adsorbent according to claim 1. 4. The adsorbent according to claim 1, wherein the ligand is selected from tert-butyl, cyclohexyl, adamantyl, norbonenyl, glycyrrhetinic acid residue, abietic acid residue and p-tert-butylphenyl group. 5. The adsorbent according to claim 1, wherein the water-insoluble resin substrate is selected from polysaccharide compounds and their derivatives, styrene resins, polyacrylamide resins and peptides. 6. The adsorbent according to claim 1, wherein the water-insoluble resin substrate is a polysaccharide compound or a derivative thereof. 7. The adsorbent according to claim 1, wherein the water-insoluble resin substrate is chitosan or a crosslinked product thereof. 8. The first claim in which the total length of the spacer group and the ligand is at least 10 angstroms.
Adsorbent according to item. 9. The total length of the spacer group and the ligand is 10
An adsorbent material according to claim 1 in the range of -60 Angstroms. 10. The adsorbent according to claim 1, which is in the form of beads. 11. A cyclodextrin-containing aqueous liquid is brought into contact with the adsorbent according to claim 1, the cyclodextrin is adsorbed on the adsorbent, and then the adsorbent is subjected to cyclodextrin desorption treatment. Separation of cyclodextrin from an aqueous liquid containing cyclodextrin, characterized by recovering cyclodextrin by
Purification method. 12. The method according to claim 11, wherein the contact is performed at room temperature. 13. The method according to claim 11, wherein the contact is continued for 1 to 4 hours. 14. The method according to claim 11, wherein the desorption treatment is performed using hot water or an alkanol / water mixed solvent. 15. A method for increasing the yield of cyclodextrin, which comprises carrying out the reaction for producing cyclodextrin by the enzymatic method in the presence of the adsorbent according to claim 1.